Project Summary The Epstein-Barr virus is a human gamma herpesvirus that infects B-lymphocytes. The EBV latent infection is associated with several human malignancies, including Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (NPC). During latency EBV is able to adopt at least four different gene expression programs that are referred to as latency types. These gene expression programs are regulated by alternative promoter utilization. Since the latency types are differentially immunogenic, the control of viral gene expression and promoter selection is a critical step for EBV to establish a long-term infection. Although progress has been made, the mechanism that regulates EBV gene expression has not yet been elucidated. The analysis of chromatin surrounding the EBV latent promoters revealed that different epigenetic patterns correspond to different latency types. Moreover, we found that the cellular factor CTCF binds at several key regions of the EBV genome and regulates promoter selection and viral gene expression. We have also proved that CTCF can promote alternative chromatin loops between the viral promoters and the origin of replication of EBV, suggesting that chromatin structure can contribute to regulate EBV latency types. This proposal, combining classical biochemical methodology and chromosome conformation analysis, seeks to reveal the role of three- dimensional architecture of the EBV genome in the regulation of viral gene expression in different latency types. The aim of this study is to use this novel approach to: 1) determine the role of CTCF/Cohesin interaction in the regulation of EBV high-order chromatin structure during latent infection; 2) determine the role of the viral protein EBNA1 in the regulation of EBV genome conformation; 3) determine if EBNA1 promotes inter- chromosomal DNA interactions between virus-host and host-host chromosomes; and 4) test if PARP1 inhibition deregulates the EBV chromosome architecture and viral gene expression. The goal of this project is to improve our understanding of the molecular mechanism that regulates EBV latency types.